The body has two systems, which are in equilibrium, in order to protect itself both from blood loss and from thromboses: the coagulation system and the fibrinolytic system. The interaction between these two systems ensures that there is initial production, for hemostasis, of insoluble fibrin polymers which are broken down again during wound healing by the lytic process of fibrinolysis.
The formation of stable thrombi as the final stage in the coagulation process is, moreover, the result of an intermeshed process which takes place like a cascade and during the progress of which each enzyme which has already been activated activates, by proteolysis, the following enzyme in the cascade and thereby amplifies the response of the body to the injury which has occurred. One of the last critical steps in this process is the polymerization of fibrin monomers. After the fibrin monomers have been formed they initially arrange themselves parallel alongside one another under the action of electrostatic forces. However, in this state they are connected only by hydrogen bonds and can be reliquefied by reagents which break hydrogen bonds, for example urea. Covalent bonding between the fibrin monomers then takes place in the presence of calcium ions by the formation of peptide linkages between the various monomers. The enzyme responsible for this network formation is activated factor XIII, called factor XIII a.
Factor XIII is a proenzyme of the blood coagulation system and can be detected both in plasma and in platelets. In plasma it occurs as a complex of "a" and "b" subunits which are not, however, covalently bonded together, whereas the form occurring in the platelets is composed only of "a" subunits. The two molecular forms of factor XIII have the same enzymatic function. After activation by thrombin and calcium, the factor XIII which has now been activated (factor XIIIa) catalyzes the formation of peptide-like linkages between particular lysine and glutamine residues in the fibrin, which results in covalent bonding of the fibrin monomers to give a network.
A genetically determined deficiency of factor XIII, or a factor XIII activation which is diminished by inhibitors, results in serious disturbances of blood coagulation. For this reason, various methods for the purification of factor XIII both from plasma and from platelets have been developed. These methods, some of which are very elaborate, are based on known protein-purification steps, for example fractional ethanol precipitation, ammonium sulfate and polyethylene glycol precipitations, as well as ion exchange chromatography or gel filtration. The authors Jan McDonagh et al. (Biochimica et Biophysica Acta, 446 (1976), 345-357) were the first to describe a method for the purification of factor XIII by affinity chromatography. They used for this purpose mercury benzoates coupled to agarose. The method provides for the reversible binding of the "a" subunit of plasma factor XIII to the mercury compound. Jan McDonagh et al. state that the "b" subunit, which itself contains no free thiol groups and thus cannot interact with the chromatography matrix, of the plasma factor remains bound to the a subunit by non-covalent forces until the latter is preactivated, by cleavage with thrombin, and is completely activated by subsequent treatment with calcium. A preparation of pure "a" subunits is thus possible by the method described by the said authors only after treatment with calcium and thrombin. Furthermore, the mercury compounds which they use are highly toxic; hence the described method cannot be used to prepare human therapeutics.